Functional Expression of Transient Receptor Potential Vanilloid 4 in Human Corneal Epithelial Cells

Abstract

Purpose:: Transient receptor potential vanilloid subtype 4 (TRPV4) is a widely expressed isoform in many different epithelia and can be activated by hypotonic stress. Such challenge to human corneal epithelial cells (HCEC) induces regulatory volume decrease (RVD) behavior. However, neither TRPV4 expression nor its role in mediating RVD has yet been identified in HCEC. In the present study, we determined whether TRPV4 channel is functionally expressed in HCEC.

Methods:: Immunostaining was used to localize TRPV4 expression in the intact human cornea and SV40-immortalized HCEC. Western Blot analysis, FACS and RT-PCR were used to detect TRPV4 protein and mRNA expression, respectively. Ca²⁺ transients were monitored in fura2-loaded HCEC using a single cell fluorescence imaging system. Relative cell volume changes were monitored in calcein-loaded HCEC using a fluorescence microplate analyzer. The Ca²⁺ transients induced by the TRPV4 agonist, 4α-PDD, and suppressed by the TRPV4 antagonist, ruthenium red (RuR), were used to validate TRPV4 function. siRNA knockdown of TRPV4 gene expression was performed with different siRNA candidates.

Results:: TRPV4 was localized in the plasma membranes of the superficial intact corneal epithelium. TRPV4 protein and mRNA expression was also identified in HCEC primary cultures and in their immortalized counterpart. FACS revealed TRPV4 delimited cell membrane expression. TRPV4-specific siRNAs suppressed TRPV4 mRNA and protein expression. Addition of 4α-PDD (3 µM) induced a 3-fold increase in Ca²⁺ influx (Δratio=0.7 ± 0.1, n=8) above its baseline value, whereas with a 50% hypotonic challenge the rise was 2.2-fold (i.e 0.6 ± 0.2, n=3) in cultured HCEC. These transients were blocked by 1 µM RuR (95%) or by a calcium free medium (97%). Following TRPV4 gene knockdown, these rises fell by 75%. In contrast, 1 µM capsazepine failed to suppress a 4α-PDD-induced transient (Δratio=0.43 ± 0.23, n=3). RuR (1 µM) or calcium free medium blocked RVD behavior by 35% (p<0.001) and 63% (p<0.001), respectively. These levels of suppression were replicated in TRPV4-siRNA transfected counterpart (33% and 49%).

Conclusions:: We determined in HCEC for the first time that there is TRPV4 functional expression. This isoform acts as an osmosensor and induces during a hypotonic challenge calcium influx and resultant RVD.

Keywords: ion channels • signal transduction • cornea: epithelium